The generation of reactive oxygen species (ROS) is inevitably linked to life. However, the precise role of ROS in signalling and specific targets are largely unknown. We performed a global proteomic analysis to delineate the yeast redoxome to a depth of more than 4,300 unique cysteine residues in over 2,200 proteins. Mapping of redox-active thiols in proteins exposed to exogenous or endogenous mitochondria-derived oxidative stress revealed ROS-sensitive sites in several components of the translation apparatus. Mitochondria are the major source of cellular ROS. We demonstrate that increased levels of intracellular ROS caused by dysfunctional mitochondria serve as a signal to attenuate global protein synthesis. Hence, we propose a universal mechanism that controls protein synthesis by inducing reversible changes in the translation machinery upon modulating the redox status of proteins involved in translation. This crosstalk between mitochondria and protein synthesis may have an important contribution to pathologies caused by dysfunctional mitochondria.

Experiment Description

We used the quantitative thiol trapping method OxICAT (Leichert et al., 2008) to determine the oxidation status of thousands of protein thiols in wild-type yeast.
For OxICAT analysis upon peroxide stress strain YPH499 was grown in three biological replicates at 28°C on minimal synthetic medium containing 2% galactose to OD600 about 0.6 and treated or not with 1 mM H2O2 for 30 min.
For OxICAT analysis of the temperature-sensitive Mia mutant mia40-4int and corresponding wild type (YPH499) strains were grown in four biological replicates at 19°C on minimal synthetic medium containing 3% [v/v] glycerol supplemented with 0.05-0.2% [v/v] glucose. Mutant and control cultures were shifted for 3 h to restrictive temperature (37°C). OD600 prior harvesting reached 0.8-1.
For mitochondria isolation, the yeast strains YPH499 or BY4741 were grown on YPG medium (1% [w/v] yeast extract, 2% [w/v] peptone, 3% [v/v] glycerol) or YPGal medium (1% [w/v] yeast extract, 2% [w/v] peptone, 2% [v/v] galactose).
Yeast cells or isolated mitochondria were pelleted, suspended in 10% TCA and immediately frozen in liquid N2. Protein extracts were prepared by bead beating in 10% TCA. Proteins were denatured in the presence of 13C-ICAT to label reduced cysteine residues. Reversibly oxidized cysteine residues were reduced by TCEP and labelled by 12C-ICAT. Proteins were digested by trypsin and ICAT labelled peptides were enriched by streptavidin affinity chromatography. Nano-HPLC-ESI-MS/MS analyses were performed on an Orbitrap Elite mass spectrometer (Thermo Scientific. OxICAT labeled samples from whole protein extracts were run twice as a technical replicate. Mass spectrometric data of OxICAT samples were processed by MaxQuant (v.1.4.1.2)(Cox and Mann, 2008) for peptide identification. MS1 ion chromatograms were extracted using Skyline (v.2.5.0)(MacLean et al., 2010) for selected peptides. Graphical displays of all chromatographic traces were visually inspected for correct peak picking. Integrated peak areas of the heavy and light version of the peptide ions were exported and the proportion of reversibly oxidized cysteine residues (%oxidation) was calculated.

Sample Description

Please note: Our data sets are from discovery-type proteomics experiments: The mass spectrometer was operated in data dependent acquisition (DDA) mode. Data is only stored on PanoramaWeb as a "Targeted MS Experiment" because it is the only option available.
The whole protein extract datasets contain two Skyline documents for each experiment ("6_20" contains peptides with a length from 6 to 21 amino acids and "21_44" contains peptides between 21 and 44 aa. For OxICAT samples of isolated mitochondria there is an additional Skyline document.
In the datasets of whole protein extracts the three biological replicates of the untreated (control) samples are named U1, U2, U3, the three biological replicates of the corresponding H2O2 treated samples are named H2O2_1, H2O2_2, H2O2_3, the four biologcal replicates of the mutant mia40-4int are named Mia1, Mia2, Mia3, Mia4 and the four biological replicates of wildtype control samples are named WT1, WT2, WT3, WT4 respectively. The endings "_TR1" or "_TR2" stand for technical replicate 1 or 2 respectively. The MitoDataset contains the data of (untreated) isolated mitochondria. "BY" stands for the strain BY4741 and "YPH" for the strain YPH499.

Site-resolved, large-scale analysis of the yeast redoxome. Yeast cells grown in 2% galactose medium were immediately frozen in 10% TCA (1). Extracted proteins were denatured in 6 M urea in the presence of heavy ICAT (13C-ICAT) to label free thiol groups (2). Reversibly oxidized cysteine residues were reduced by TCEP and labeled by light ICAT (12C-ICAT) (3). Proteins were digested with trypsin and ICAT-labeled peptides were enriched by streptavidin affinity chromatography (4). Following cleavage of the biotin tag, peptides were analyzed in duplicate by LC-MS/MS. (b) For the accurate determination of the redox state of cysteine residues, peptides were identified based on fragment ions observed in MS2 spectra using MaxQuant (v.1.4.1.2) and then quantified by extracting MS1 ion chromatograms using Skyline (v.2.5.0). Following integration of peak areas of heavy and light peptide variants, the proportion of reversibly oxidized cysteine residues (% oxidation) was calculated.

Please note: When the retention time profiles of “light” and “heavy” peaks were inconsistent, the respective XICs were excluded from quantification. The information which XICs were not quantified can be found in the tables below (denoted by “nq”).

Table S2. Oxidation status of cysteine containing peptides of non-stressed yeast cells. MS1 ion chromatograms were extracted using Skyline (v.2.5.0). Integrated peak areas of the “heavy” (13C-ICAT labelled) and “light” (12C-ICAT labelled) version of the peptide ions were exported and the proportion of reversibly oxidized (12C-ICAT labelled) cysteine residues (%oxidation) was calculated. The table contains all cysteine containing peptide sequences quantified in Skyline in at least two of three biological replicates.